Therapeutic serum obtained from co-cultured cells

ABSTRACT

A therapeutic serum suitable for inclusion in a cosmetic preparation may be produced by stressing a co-culture including proliferative cells. The co-culture of cells may be obtained by growing first culture to less than one-hundred percent confluence on a surface. After a monolayer of first culture is established, a second culture may be seeded onto at least one cell free area on the surface, the resulting co-culture grown to less than one-hundred percent confluence. Additional cultures may then be seeded onto cell free areas of the surface and established until a monolayer having the desired population of cells is obtained. The monolayer is then stressed to obtain a serum by conditioning a collection medium. The obtained serum may be combined with a suitable cosmetic base to provide a cosmetic preparation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a continuation of U.S. application Ser. No.15/943,320, filed Apr. 2, 2018, the teachings of which are herebyincorporated by reference in their entirety, which is acontinuation-in-part of U.S. application Ser. No. 15/700,895, filed Sep.11, 2017, the teachings of which are hereby incorporated by reference intheir entirety, which is a continuation-in-part of U.S. application Ser.No. 14/597,796, filed Jan. 15, 2015, the teachings of which are herebyincorporated by reference in their entirety, which claims priority toProvisional U.S. Application No. 61/927,674, filed Jan. 15 2014, theteachings of which are hereby incorporated by reference in theirentirety.

FIELD

A therapeutic serum, a method of obtaining such serum, and medicaldevices and preparations containing such serum are provided.

BACKGROUND

The various organs of the body are composed of multiple cell typesworking together. By working together, the different cell types maintainthe health the organ and help the organ recover from injury. Consider,for instance, the largest organ of the body, skin.

Skin is an essential multilayer organ. Providing a barrier againstpathogens and toxins, as well as synthesizing nutrients such as vitaminD, skin is essential for maintaining an individual's physical health.The integrity of skin is also essential for maintaining one'spsychological health. Skin is the most prominent part of an individual'sbody. Blemishes, scars, wrinkles and perceived imperfections candiminish an individual's self-confidence. Maintaining physical andpsychological health, therefore, requires maintaining healthy skin.

Healthy skin comprises layers of different cells supported by ascaffolding of proteins called the extracellular matrix. Theextracellular matrix supporting the skin comprises various proteins suchas collagen, fibronectin and laminin. These and other structuralproteins intertwine and communicate to form the structural and dynamicthree-dimensional scaffolding providing skin with its strength andresilience. Cells forming the various layers of skin adhere to theextracellular matrix, and rely on both structural and biologicallyactive signal relaying molecules within the matrix to maintain properfunction. During wound healing, cells also use the extracellular matrixas a bridge to migrate into and close wounds. Providing support andpathways for healing, the extracellular matrix is an importantstructural and biologically active component of healthy skin. As anindividual ages, however, the extracellular matrix changes and becomesweakened, leading to the appearance of wrinkles, blemishes and decreasedhealing.

The extracellular matrix is manufactured and maintained primarily byfibroblasts beneath the skin's surface. Manufacturing and maintainingthe biological activity of the extracellular matrix, healthy fibroblastsare essential for healthy skin. Above the fibroblasts, on top of theextracellular matrix, are keratinocytes forming the epidermis. Formingthe epidermis, the outer layer of skin, healthy keratinocytes are alsoessential for the appearance and integrity of skin. Signaling betweenkeratinocytes and immune cells helps to maintain homeostasis of the skinand protect against infections. Accordingly, maintaining healthy skinfrom its base to its surface requires promoting the health of differentcells throughout the layers of skins.

The same is true for other organs of the body. The nervous system, forinstance, is composed of various neurons and glial cells. Carryingsignals to and from the brain, as well as processing information withinthe spinal cord and brain, neurons are often thought of as the primaryworkhorses of the nervous system. Neurons, however, are supported byvarious glial cells. For instance, astrocytes maintain the health ofneurons and help the brain recovery from various injuries, such asstroke. Schwan cells insulate peripheral nerves and help them regenerateafter injury. Accordingly, just like the skin, the health and healing ofthe nervous system requires the promoting the health of different cells.

The liver, another important organ of the body, also relies on multiplecell types to maintain health and healing. Repairing and maintaining theextracellular matrix, stellate cells, for instance, are important formaintaining health and healing.

SUMMARY

A serum collected from a growing co-culture of cells may provide thevarious proteins, cytokines, glycans, hormones and other molecularfactors necessary for promoting the health of and healing various organsof the body. Given the importance of the extracellular matrix in organs,such may be accomplished by remolding and maintenance of theextracellular matrix, as to promote wound healing, promote homeostasis,help maintain the integrity of the organ and/or scars. Given that organsare composed of different cell types acting in concert, collecting aserum from a co-culture comprising various cells types would bettermatch the complete needs of injured and/or aging organs. Healthy skin,for instance, comprises various cells, such as keratinocytes,fibroblasts, mesodermal cells, melanocytes, Merkel Cells, Langerhanscells, etc., collecting a serum from a growing co-culture comprisingkeratinocytes, fibroblasts, mesodermal cells, melanocytes, Merkel Cells,Langerhans cells, T-cells and/or other skin cells in variouscombinations would better match the complete needs of new and agingskin. Similarly, collecting a serum from a co-culture of hepatocytes,stellates, Kupffer cells and/or endothelial cells would better match theneeds of injured, diseased and/or aging livers. Collecting a serum froma co-culture of comprising all or a portion of the various cells withinnervous system, likewise, may assist the nervous system in recoveringfrom injury and age. However, different growth rates and/or nutrientsrequirements may complicate efforts to co-culture different cells. Aportion of the cells to be co-cultured, for instance, may only be ableto survive in a growth medium that promotes the proliferation of othercells within the co-culture. With some cells proliferating and othersonly surviving, the cultures could be become dominated by theproliferating cells. That is, the resulting co-culture may comprisedisproportionate amounts of the various cell types. Dominated by onetype of cell over the other types, intercellular signaling, such asparacrine signaling, may be altered, diminished and/or lost. Havingaltered and/or diminished intercellular signaling, such a culture wouldbe unlikely to produce a serum having all the proteins and/or othermolecular factors necessary for healing and promoting the health ofinjured and/or aging organs.

Dominance by one cell type, i.e. a disproportionate amount, within aco-culture may be avoided by seeding a first surface with a firstculture of cells and allowing the culture to become established bygrowing the first culture to a monolayer of less than one-hundredpercent confluence. Growing the first culture to a monolayer of lessthan one-hundred percent confluence may be facilitated by growing thefirst culture in the presence of a growth medium comprising nutrientsand at least one growth factor. The first growth medium should promotethe proliferation of the first cell culture. Growing the first cultureto less than one-hundred percent confluence provides at least one cellfree area on the first surface. A second culture of cells may then beseeded onto at least a portion of the cell free areas of the firstsurface. The second culture of cells may comprise cells different thanthe first culture. After being seeded onto at least one of the cell freesurface areas of the first surface, the second culture and first culturecan be grown to less than one-hundred percent confluence in the presenceof a second growth medium. The second growth medium should promote theproliferation of the second culture and may comprise nutrients and atleast one growth factor. Depending upon the cells within the first andsecond cultures, the first growth medium and second growth medium may beidentical, include shared components and/or be completely different.Accordingly, the second growth medium may comprise the nutrients and atleast one growth factor of the first growth medium. The procedure may berepeated with subsequent cell cultures and subsequent growth medium,which may allow for complex and/or diverse co-cultures. Following thegeneral procedure of seeding subsequent cultures onto cell free areas ofa surface may facilitate establishment of a co-culture by providing thesubsequent cultures room and/or favorable conditions to becomeestablished. Favorable conditions may be provided by changing the growthmedium and/or other conditions, such as temperature and/or pH, as topromote proliferation of the subsequently seeded cultures. Favorableconditions promoting the proliferation of subsequently seeded culturesshould not be such as to cause previously seeded cultures to becomesenescent. The serum collected from a co-culture of cells may beenhanced by maintaining the cells in a proliferative phase by growingthe final co-culture to less than 100% confluence on the first surfaceafter seeding the final culture to be added. Maintaining the cells in aproliferative phase prior to and/or during serum collection mayinfluence the composition of the serum.

Production of the serum may be induced in a co-culture by replacing thefirst and/or second growth medium with a collection medium lacking allor a portion of the growth factors of the final growth medium, after thefinal co-culture has been grown to less than one-hundred percentconfluence. For instance, the final growth medium may be replaced with acollection medium lacking at least one of the growth factors of thesecond growth medium. The collection medium may comprise the nutrientsof at least one of the first and second growth mediums. The co-culturemay then be maintained for a period of time in the collection mediumsufficient to allow the co-culture to produce from the collection mediuma conditioned medium comprising the therapeutic serum. After which, atleast a portion of the conditioned medium containing the therapeuticserum may be collected.

Facilitating growth of a monolayer may be accomplished by utilizing asurface enabling growth of the co-culture radially outwards as a singlelayer from each seed cell and/or collection of cells. Seeding a surfacecomprises depositing cells of the culture to be grown onto the surface.Depending on the manner utilized to deposit cells onto the surface, thecells may be deposited as individuals or clusters. For instance, seedingthe cells from a solution may cause individual cells to be depositedover the area of the surface seeded. In combination or the alternative,the surface may be seeded by depositing tissue samples and/or portionsof a previously grown culture onto the surface. In such instances,clusters of cells may be deposited onto the area of the surface seeded.The surface should allow the culture to grow radially outwards fromdeposited cells. Of course, portions of the culture growing radiallyoutward from individually seeded cells and/or clusters may mergetogether. The cells may also coalesce together to form variousthree-dimensional configurations. Hepatocytes, for instance, mayself-assemble into spheroids. Accordingly, a monolayer may comprisevarious three-dimensional aggregates, provided that such aggregates andother cells within the co-culture are predominately arranged as a singlelayer.

Radial growth from seeded cells and/or clusters providing a monolayermay be facilitated with a variety of surfaces. For instance, the bottomof Erlenmeyer flask and/or petri dish may provide a sufficient surface.The sides of T-flask may also provide a sufficient surface. When vesselssuch as a petri dishes and/or flasks are utilized, they should be sizedto provide a sufficient volume of growth medium enabling growth of thefinal co-culture to be obtained.

The surface does not need to be the sides of a vessel. For instance, thesurface may be one or more plates submerged in a growth medium.

The surface may also be provided by a gelling agent, such as agar. Insuch instances, the culture could be grown on the solidified gellingagent. The gelling agent may include all or a portion of the growthmedium. When the gelling agent does not include all of the growthmedium, the missing elements of the growth medium may be provided bysubmerging the solidified gelling agent in an appropriate solution.

As to facilitate growth and establishment, a first and/or subsequentculture may be provided with a growth medium comprising nutrients andgrowth factors. Growth factors are substances capable of stimulatinghealing, growth, cellular proliferation and/or cellular differentiation.Growth factors useful for facilitating growth and establishment of aculture of skin cells may include, but are not limited to, amino acids,such as L-Glutamine, hormones, such as hydrocortisone hemisuccinate,insulin and/or epinephrine, omega fatty acids, such as linoleic acid,vitamins, such as vitamin C, proteins, such as serum albumin, basicfibroblasts growth factor, acidic fibroblasts epidermal growth factor,transforming growth factor, insulin, bovine pituitary extract and/orApoTransferin, and/or glycerophospholipids, such as lecithin. Othergrowth factors may facilitate the growth of the other cells to beco-cultured. Accordingly, the growth factors added may be chosen topromote the growth and/or proliferation of a culture.

The nutrients in the growth medium provided to a culture need not belavish or exceed the minimal nutrients required for survival and growthof the culture. As such, a basal medium and/or minimal essential mediumcan provide sufficient nutrients. The nutrients of the first, secondand/or other subsequent growth medium, therefore, may be provided by abasal medium. Accordingly, the first, second and/or other subsequentgrowth medium provided to a culture may comprise growth factors combinedwith a basal medium and/or minimal essential medium. The growth mediumprovided to a first, second and/or other subsequent cultures may includea balancing agent to buffer the medium to a desired pH, such as, but notlimited to, Earl's salts and/or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

Accordingly, when establishing a co-culture of cells, a first seededculture may be provided with a growth medium comprising nutrients and atleast one growth factors as to facilitate establishment of the cultureafter being seeded onto a surface. The growth medium provided to thefirst seeded culture may comprise a basal medium and/or minimalessential medium. The growth factors included within the growth mediumprovided to the first and/or subsequent seeded cultures may include, butare not limited to, amino acids, such as L-Glutamine, hormones, such ashydrocortisone hemisuccinate, insulin and/or epinephrine, omega fattyacids, such as linoleic acid, vitamins, such as vitamin C, proteins,such as serum albumin, basic fibroblasts growth factor, epidermal growthfactor, transforming growth factor, insulin, bovine pituitary extractand/or ApoTransferin, and/or glycerophospholipids, such as lecithin.

Different growth rates and/or nutrients requirements may complicateefforts to co-culture different cells. Keratinocytes, for instance, mayonly be able to survive in a growth medium that promotes theproliferation of T cells. Consequently, attempting to co-culture T-cellswith keratinocytes and/or other skin cells can easily result in aculture in which not all cell types are proliferating. With some celltypes proliferating and others only surviving, the cultures could bebecome dominated by the proliferating cells. Being dominated by one typeof cell over the other types, intercellular signaling, such as paracrinesignaling, may be altered, diminished and/or lost. Having altered and/ordiminished intercellular signaling, such a culture would be unlikely toproduce a serum having all the proteins and/or other molecular factorsnecessary for healing and promoting the health of wounded and/or agingskin.

For instance, traditional keratinocyte mediums, such as Dulbecco'sModified Eagle Media (DMEM), Keratinocyte Growth Medium (KGM, Lonza) andRoswell Park Memorial Institute medium (RMI medium), are effective forpromoting the proliferation of keratinocytes. However, such mediums maynot sufficiently promote the growth and survival and T-cells. Theresulting co-culture, accordingly, may be dominated by keratinocytes.Attempting to co-culture keratinocytes and T-cells in a growth mediumpromoting the survival and growth of T-cells, such as Xvivo15 fromLonza, may not promote the proliferation of keratinocytes. Utilizing amedium not promoting the proliferation of keratinocytes, the resultingculture may be dominated by T-cells. Dominated by keratinocytes orT-cells, the natural paracrine signaling between the cells may be lost.Paracrine signaling between keratinocytes and T-cells may stimulate theproduction of molecular factors, such as cytokines like IL-17, usefulfor promoting homeostasis of the skin and defending against fungal andbacterial infections. A serum comprising such homeostasis and/ordefensive molecular factors may be beneficial in promoting the health ofskin and/or other organs. In combination or the alternative, a serumcomprising such homeostasis and/or defensive molecular factors may bebeneficial in protecting organs from infection. Co-cultures dominated bykeratinocytes or T-cells may not provide such beneficial serums due tointercellular signaling, such as paracrine signaling, being altered,diminished and/or lost.

Dominance by one cell type within a co-culture may be avoided by seedinga surface with a first culture and growing the first culture to amonolayer of less than one-hundred percent confluence in the presence ofa first growth medium comprising nutrients and at least one growthfactor, as to provide at least one cell free area. All or a portion ofthe cell free areas of the first surface may then be seeded with asecond culture. After seeding of the second culture, the first andsecond cultures may be grown to a monolayer of less than one-hundredpercent confluence in the presence of a second growth medium comprisingnutrients and at least one growth factor. The second growth mediumshould promote proliferation of the second culture. Accordingly, in thecase of a co-culture of T-cells and keratinocytes, the first culture maycomprise keratinocytes and the first growth medium may comprise a growthmedium promoting the proliferation of the keratinocytes, such as DMEM,KGM or RMI medium. After the keratinocytes have been grown to asufficient monolayer, the first surface may be washed to remove thefirst growth medium. All or a portion of the cell free areas on thefirst surface may then be seeded with a second culture comprisingT-cells. The second and first culture may then be grown to a monolayerof less than one-hundred percent confluence in the presence of a secondgrowth medium promoting the proliferation of T-cells, such as Xvivo15.As to maintain at least some level of proliferation of the firstculture, the second medium may comprise at least one growth factorfacilitating and/or promoting the proliferation of the first cultureand/or intervening cultures. For instance, if the first culturecomprises keratinocytes, the second growth medium may comprise at leastone of bovine pituitary extract, L-glutamine, hydrocortisonehemisuccinate, transforming growth factor, insulin, epinephrine, andApoTransferrin, or combinations thereof.

Different responses to growth factors may also complicate efforts toco-culture different types of cells. For instance, hepatocytes canspontaneously form spheroids when cultured. The formation of spheroids,however, is inhibited by fetal bovine serum. Obtaining a monolayercomprising hepatocytes spheroids, accordingly, requires limiting and/orprecluding exposure to fetal bovine serum. Stellates, another cell ofthe liver, require the presence of FBS to grow in culture. Providing asecond culture comprising hepatocyte spheroids after a first culture ofstellates may require replacing a first growth medium comprising FBSwith a second growth medium lacking FBS. The second growth medium,therefore, may lack a nutrient and growth factor of the first growthmedium.

Attempts to co-culture different types of cells may also be complicatedby different growth rates in response to the growth medium. A growthmedium including basic fibroblast growth factor (bFGF) stimulatesproliferation of fibroblasts more than keratinocytes. Conversely, acidicFGF stimulates proliferation of keratinocytes more than fibroblasts. Assuch, an acidic growth medium may cause a co-culture of fibroblast andkeratinocytes to be dominated by keratinocytes, whereas a basic growthmedium may cause the co-culture to be dominated by fibroblast. Beingdominated by fibroblast over keratinocytes, or vice versa, intercellularsignaling, such as paracrine signaling, may be altered, diminishedand/or lost. Having altered and/or diminished intercellular signaling,such a culture would be unlikely to produce a serum having all theproteins and/or other molecular factors necessary for healing and/orpromoting the health of injured and/or aging organs.

Dominance by one cell type over another due to different responses togrowth factors may be lessened and/or avoided by removing growth factorsfavoring proliferation of previous seeded cultures prior to growinglater seeded cultures. In combination or the alternative, the growthfactors favoring the later seeded culture may be added to the growthmedium. Accordingly, the first growth medium may comprise a growthfactor promoting the proliferation of a first seeded culture. In someinstances, the first growth medium may comprise growth factors promotingthe proliferation of the first culture more than subsequent cultures.For instance, if the first culture comprises fibroblast cells, the firstgrowth medium may comprise bFGF. After the first seeded culture has beengrown to a monolayer of less than one-hundred percent confluence, as toprovide at least one cell free area, the first surface may be washed toremove the first growth medium. A second cell culture may then be seededonto all or a portion of the cell free area. The first and secondcultures may then be grown to a monolayer of less than one-hundredpercent confluence in the presence of a second growth medium. The secondgrowth medium may comprise a growth factor promoting the proliferationof cells within the second culture more than cells within the firstculture. For instance, if the second culture comprise keratinocytes, thesecond growth medium may comprise aFGF. As to maintain at least somelevel of proliferation of the first culture, the second medium maycomprise at least one growth factor facilitating and/or promoting theproliferation of the first culture and/or intervening cultures.

Attempts to co-culture different types of cells may also be complicatedby different growth rates of the cells, regardless of the growth medium.Some cells of the intended co-culture may be slow to proliferate withrespect to other cells of the co-culture across many different growthmediums. Growing quicker than other cells within the co-culture, thefaster proliferating cells may crowd out the other cells within theco-culture. Consequently, attempting to co-culture faster proliferatingcells with slower proliferating cells may result in a co-culturedominated by the faster proliferating cells over the slowerproliferating cells.

Dominance by one cell type over another due to different growth ratesmay be lessened and/or avoided by allowing slower growing cells tocreate a sufficient monolayer on a surface prior to seeding fastergrowing cells onto the surface. For instance, the first seeded culturemay comprise cells with slower doubling times than other cells to becultured. After the first seeded culture has been sufficientlyestablished on the surface, cultures comprising more proliferative cellscan be seeded onto at least one cell free area on the surface to providea co-culture. Once the more proliferative cells have been established,subsequent cultures can be seeded and established until a co-culturehaving the desired cellular composition is obtained on the surface.Achieving the desired cellular composition of the final co-culture mayrequire seeding successive cultures onto the surface at varyingconfluences. For example, if the final co-culture is to comprise aco-culture of a first cell type, a second cell type with faster growthrate than the first cell type, and a third cell type with a fastergrowth rate than the first and second cell types, then it may beappropriate to first seed a culture of the slowest growing cells ontothe surface and grow the first seeded culture to approximatelythirty-three percent confluence. A culture of cells having theintermediate growth rate may then be seeded onto at least one cell freearea on the first surface. The co-culture of the first and second celltypes may then be grown to approximately sixty-six percent confluence.Then a culture of the fastest growing cell types may be seeded onto atleast one cell free area on the first surface and the co-culture of thethree cells grown to less than one-hundred percent confluence.

The above examples are based upon situations in which subsequent seededcultures comprise cells growing sufficiently faster than previouslyseeded cultures such that the growth of previously seeded cultures canbe treated as halted, and that a final co-culture having equal amountsof each culture is desired. One or both these assumptions may not betrue for every co-culture to be produced. Accordingly, it may beadvantageous to grow the first seeded culture and/or subsequentco-cultures to other confluences as to account for growth rates ofpreviously seeded cultures with subsequently seeded cultures and/or thedesired cellular composition of the final co-culture.

The composition of serum generated by a co-culture of cells may also bealtered by cells becoming senescent. Cells are more productive when theyare actively proliferating. Additionally, cells in the proliferativephase produce different molecular factors then senescent cells. Havingaltered and/or diminished production of molecular factors, a co-culturecomprising senescent cells may also be unlikely to produce a serumhaving all the proteins and/or other molecular factors necessary forhealing and/or promoting the health of injured and/or aging organs.Preserving intercellular signaling and/or maintaining cells in theproliferative phase in a co-culture of cells, accordingly, may provide atherapeutic serum suitable for promoting the health and/or healing ofvarious organs. Therefore, it may be desirable to maintain cultures fromwhich cells are to be harvested for co-culturing in a proliferativephase. Likewise, it may be desirable to maintain cells within seededcultures in a proliferative phase.

Maintaining cells within a culture in a proliferative phase may beaccomplished by preventing the culture form achieving one-hundredpercent confluence. Accordingly, as to maintain the cells of the firstseeded culture in a proliferative phase, the first seeded culture shouldbe grown to a monolayer of less than one-hundred percent confluence, asto provide at least one cell free area on the surface. After the firstculture has been established, a second culture may be seeded onto all ora portion of the cell free areas. The first and second culture may thenbe grown to less than one hundred percent confluence.

The cells of the second or other subsequent cultures seeded onto thesurface having a monolayer of preceding cultures may be provided insuspension. The suspension may be acquired from one or more separatelygrown cultures. Such separate cultures may be grown on a surface and/orin suspension in the presence of a growth medium including growthfactors and nutrients. The nutrients provided in the growth medium ofthe cultures from which cells to be seeded are harvested do not need tobe lavish or exceed the minimal nutrients required for survival andgrowth of the culture. As such, a basal medium and/or minimal essentialmedium can provide sufficient nutrients. Accordingly, the growth mediumprovided to cultures from which cells are harvested may comprise growthfactors combined with a basal medium and/or minimal essential medium.The growth medium provided to subsequent cultures may include abalancing agent to buffer the medium to a desired pH, such as, but notlimited to, Earl's salts and/or HEPES.

Growth of subsequent cultures with the first culture after seeding maybe facilitated and/or enhanced by maintaining the cells of subsequentcultures in a proliferative phase. Cells of subsequent cultures may bemaintained in a proliferative phase by growing the cells on a secondsurface to less than one-hundred percent confluence prior to seedingonto the first surface of the co-culture. For instance, growing thecells of subsequent cultures on a second surface to approximately 80 to90 percent confluence before seeding onto the surface of the co-culturemay maintain the cells of the subsequent culture in a proliferativephase prior to seeding.

As to provide subsequent cultures a sufficient surface to becomeestablished, the first culture and/or preceding co-cultures are grown tomonolayers of less than one-hundred percent confluence. For instance,the first culture and/or proceeding co-cultures may be grown to amonolayer of approximately eighty to ninety percent confluence, beforeseeding the second and/or subsequent cultures onto the at least one ofthe cell free areas of the first surface. Though previous cultures aregrown to less than one-hundred percent confluence, it may be necessaryto provide areas on the surface free of preceding cultures. Providingsuch surfaces within a monolayer of established cultures may beaccomplished by growing preceding cultures to a monolayer of less thanone-hundred percent confluence and then increasing the cell free surfacearea by creating voids in the monolayer of the first culture and/orpreceding co-cultures by removing an appropriate amount of themonolayer. The amount removed will be dependent on the growth rate ofthe cells together and/or the final cellular composition of theco-culture desired. For example, if a co-culture comprisingapproximately equally amounts of two cell types is desired, and thesecond seeded cell type is sufficiently aggressively as to fill voidswithout allowing the first cells to significantly enter the voids, thenapproximately 50% of the monolayer of the first culture may be removed.

Voids may be created by removing portions of the monolayer of precedingcultures from the surface. For instance, voids may be created byscraping or otherwise mechanically detaching portions of the monolayerof the first culture and/or preceding co-cultures from the surface. Itis also possible to remove portions of an established monolayer by firsttreating the monolayer with a detachment solution for a sufficient timeto cause cells in the monolayer to begin to ball. Once the cells of themonolayer begin to ball, the detachment solution can be withdrawn andsquirted back onto the monolayer to produce voids in the monolayer.

The serum collected from a co-culture of cells may be enhanced bymaintaining the cells in a proliferative phase by growing the finalco-culture to less than one-hundred percent confluence on the firstsurface after seeding the final culture to be added. Maintaining thecells in a proliferative phase prior to and/or during serum collectionmay influence the composition of the serum. The composition of the serummay be adjusted by allowing at least a portion of the cells to senesce.Accordingly, the composition and/or ratio of senesced versusproliferative cells within the co-culture may influence the compositionand/or quality of the serum collected.

The quality of the serum may also be enhanced by stressing the cellswithin the co-culture. Cells within the co-culture may be stressed byselectively depriving the cells of at least one growth factor, nutrientand/or metabolic component. For example, cells within the co-culture maybe stressed by depriving the cells of one or more growth factors whilemaintaining nutrient levels. Cells within the co-culture may by stressedafter the co-culture has grown to approximately 80 to 95% confluence orat other times. Accordingly, production of a therapeutic serum may beinduced in a co-culture by replacing the first and/or second growthmedium with a collection medium lacking all or a portion of the growthfactors of the final growth medium, after the final co-culture has beengrown to less than one-hundred percent confluence. For instance, thesecond growth medium may be replaced with a collection medium lacking atleast one of the growth factors of the second growth medium. Thecollection medium may comprise the nutrients of at least one of thefirst and second growth mediums. The co-culture may then be maintainedfor a period of time in the collection medium sufficient to allow theco-culture to produce from the collection medium a conditioned mediumcomprising the therapeutic serum. After which, at least a portion of theconditioned medium containing the therapeutic serum may be collected.

As to facilitate further production of the therapeutic serum, thewithdrawn portion of the conditioned medium may be replaced with freshcollection medium. Production of the therapeutic serum may also beenhanced by allowing the co-culture to recover from the induced stress.Such a recovery phase may be provided by replacing the conditionedmedium with a recovery medium containing the removed growth factors andculturing the co-culture in the recover medium for a recovery period oftime. Preventing the co-culture from obtaining one-hundred percentconfluence may improve and/or maintain the quality of the serumproduced. The recovery medium may comprise the growth factors and/ornutrients of at least one of the first, second and other growth mediumutilized in establishing the co-culture. Depending on the cells includedwithin the co-culture, a recovery period of approximately 24 to 72 hoursmay be sufficient.

The collected therapeutic serum may be applied to directly an organ. Forinstance, the serum may be sprayed onto exposed organs, such as skin, ororgans exposed during surgery and/or injury. The therapeutic serum mayalso be injected into and/or around the organ intended for treatment.

The serum may also be incorporated into a delivery medium. For instance,after the therapeutic serum has been collected, a delivery mediumcomprising a cosmetic preparation may be produced by adding thetherapeutic serum to a cosmetically suitable base. Accordingly, acosmetic preparation may comprise a serum prepared by any of the abovedetailed processes and a cosmetic base. The cosmetic base which may beused is not particularly limited and may comprise at least one of ahydrogel, such as polyethylene glycol, an oil, such as sunflower seedoil, sweet almond oil and/or coconut oil, and/or a fat, such as SheaButter, or various combinations thereof. The cosmetic base may alsoinclude alcohols, polyols, emulsifiers, such as Ceteareth-20, carbomerand/or glycerol monostearate, preservatives, and/or moisturizers, suchas hyaluronic acid. An Antioxidant, such as vitamin E, vitamin A and/orvitamin C may also be included in the composition. The cosmeticpreparation may include phytochemicals, such as resveritol, quercetinand/or epigallocatechin gallate. If desired, a fragrance may also beadded to the cosmetic preparation. The cosmetic preparation may containother ingredients, such as pigments, flavoring agents, preservativesand/or sweeteners. The ingredients included within the cosmeticpreparation are not particularly limited, as long as they collectivelyprovide a cosmetically suitable preparation that is non-toxic whentopically applied.

The delivery medium may comprise a physical structure such as a gauze,mesh, bandage and/or dressing. For instance, a serum derived from skincells for treating wounds may be incorporated into a wound dressing,such as silicone mesh, dressing, bandage or gauze. The wound dressingshould be sufficiently porous and/or have sufficient internal spaces asto hold and elude a therapeutically effective amount of the serum.

The physical structure may also comprise an implantable structure, suchas a biocompatible mesh. The mesh may be placed over, adjacent and/orwithin the organ to be treated. For instance, a serum derived from aco-culture cells of liver cells may be placed in proximity to the liver.The implantable structure should be sufficiently porous and/or havesufficient internal spaces as to hold and elude a therapeuticallyeffective amount of the serum. The implantable structure may bebioabsorbable as eliminate the need for subsequent surgeries to retrievethe structure. Additionally, absorption of the structure by the body mayfacilitate release of a therapeutic effective amount of the serum overtime. Appropriate bioabsorbable structures may be fabricated frompoly(hydroxyvalerate), poly(L-lactic acid), polcaprolactone,poly(lactide-co-glycolide), poly(hydroxybutyrate),poly(hydroxybutyrate-co-valerate), polydioxanone, polyorthoesters,polyanhydrides, poly(glycolic acid), poly(D.L-lactic acid),poly(glycolic acid-co-trimethylene carbonate), polyphosphoesters,polyphosphoester urethanes, poly(amino acids), cyanoacrylates,poly(trimethylene carbonate), poly(iminocarbonate), copoly(ether-esters)(e.g. PEO/PLA), polyalkylene oxalates, polyphosphaZenes and biomoleculessuch as fibrin, fibrinogen, cellulose, starch, collagen, hyaluronicacid, etc., and mixtures thereof.

The delivery medium may also comprise a graft in which the therapeuticserum is infused. For instance, the therapeutic serum may be providedwithin a skin graft and/or vascular graft. The graft may be supported bya matrix. Matrixes supporting the graft may be composed of anybiocompatible material. In some instances, the matrix supporting thegraft may comprise bioabsorable materials, such aspoly(hydroxyvalerate), poly(L-lactic acid), polcaprolactone,poly(lactide-co-glycolide), poly(hydroxybutyrate),poly(hydroxybutyrate-co-valerate), polydioxanone, polyorthoesters,polyanhydrides, poly(glycolic acid), poly(D.L-lactic acid),poly(glycolic acid-co-trimethylene carbonate), polyphosphoesters,polyphosphoester urethanes, poly(amino acids), cyanoacrylates,poly(trimethylene carbonate), poly(iminocarbonate), copoly(ether-esters)(e.g. PEO/PLA), polyalkylene oxalates, polyphosphaZenes and biomoleculessuch as fibrin, fibrinogen, cellulose, starch, collagen, hyaluronicacid, etc., and mixtures thereof. In some instance, the graft may onlycomprise the matrix.

As to better match the individual to be treated, the co-culture of cellsproviding the therapeutic serum may be harvested from the patient to betreated and/or related individual.

DETAILED DESCRIPTION

A method for obtaining a therapeutic serum suitable for inclusion in nowwill be described more fully with reference to specific examples. Theserum, however, may be obtained in different manners, and thus shouldnot be construed as limited to the specific examples provided.Accordingly, the serum may be obtained by a different ordering and/orsequence of the various steps and/or procedures detailed in the providedexamples. For example, two or more steps may be performed concurrentlyor with partial concurrence. Also, some steps that are performed asdiscrete steps in the following examples may be combined, and stepsbeing performed as a combined step may be separated into discrete steps,the sequence of certain steps may be reversed or otherwise varied, andthe nature or number of discrete steps may be altered or varied.Accordingly, the provided examples are not intended to exclude any ofsuch means of obtaining a therapeutic serum suitable for use in acosmetic preparation.

Likewise, different reagents, techniques, materials and/or equipmentother than those specifically mentioned may be utilized to provide thetherapeutic serum.

A therapeutic serum may be produced by stressing a co-culture includingproliferative cells. The co-culture of cells may be obtained by firstestablishing a monolayer of a first cell culture on a surface. After amonolayer of a first culture is established, a second culture may thenbe seeded onto cell free areas within the monolayer and established.Additional cultures may then be seeded and established until a monolayerthe having the desired cellular composition is obtained. The monolayerof the final co-culture is then stressed to obtain a serum byconditioning with a collection medium. The obtained serum may beincorporated into a suitable delivery medium.

For instance, a serum comprising Human Neonatal Fibroblast/KeratinocyteConditioned Media may be obtained from a proliferative monolayercomprising a co-culture of keratinocytes and fibroblasts. Theco-cultured monolayer may be established by first partially submerging avial of frozen keratinocytes (obtained from LifeLine Cell Technologies)in a 37° C. water bath, without submerging the top of the vial. The vialis allowed to thaw in the water bath until a small piece of ice remains.The vial is then removed and sprayed with an ethanol solution. In ahood, keratinocytes are seeded from the vial at 2,500 to 5,000 cells percm² onto a culture treated surface. The surface should be provided withan appropriate volume of a suitable growth medium, such as a mediumincluding Basal DermaLife Media (LifeLine Cell Technologies) and growthfactors comprising bovine pituitary extract, L-glutamine, hydrocortisonehemisuccinate, transforming growth factor, insulin, epinephrine and/orApoTransferrin. The seeded surface is then placed in an incubator andgrown at 37° C. in the presence of humidified air comprising 5% CO₂. Asto remove any residue DMSO and/or other solvents that may be present inthe cryogenic solution, the growth medium may be changed every 24 to 48hours following initiation of the monoculture. After which time, thegrowth medium may be changed every 48 to 72 hours.

Other means of obtaining the initial keratinocytes may also be employed.For instance, keratinocytes may be isolated from neonatal foreskinretrieved from circumcision using the techniques detailed in U.S.application Ser. No. 14/597,796, filed Jan. 15, 2015, the teachings ofwhich are hereby incorporated by reference in their entirety.

The keratinocytes are allowed to grow in the growth medium until 80-90%confluence is achieved. Voids are then created within the establishedmonolayer by removing the growth medium and washing twice with anappropriate volume of a buffer solution, such as phosphate buffersolution without calcium or magnesium. After washing with buffersolution, a sufficient volume of an enzymatic cell detachment solutionto promote detachment of the cells from the surface is added. The celldetachment solution may comprise proteolytic and/or collagenolyticenzymes. For instance, detachment of the cells may be promoted by adding1 ml of HyQTase Solution, manufactured by HyClone Laboratories, Inc, per25 cm² of growth area. The enzymatically treated monolayer may then beincubated at 37° C. until the keratinocytes start balling. The surfaceis then tilted to collect the enzyme solution with a pipette. Thecollected solution is sprayed at focused points onto the monolayer tocreate voids in about 50% of the monolayer. The enzyme solution anddetached keratinocytes are then removed. The remaining monolayer isprovided with a sufficient volume of the growth medium and returned tothe incubator. For example, an amount of medium providing 10 ml ofmedium per 55 cm² of growth area may be sufficient.

Simultaneously, a monolayer of fibroblasts is cultured on a secondsurface by submerging a vial of frozen fibroblasts (obtained fromLifeLine Cell Technologies) in a 37° C. water bath, without submergingthe top of the vial. The vial is allowed to thaw in the water bath untila small piece of ice remains. The vial is then removed and sprayed withan ethanol solution. In a hood, fibroblasts are seeded from the vial at2,500 to 5,000 cells per cm² on to a culture treated surface. Thesurface should be provided with an appropriate volume of a suitablegrowth medium, such as a medium including Basal DermaLife Media(LifeLine Cell Technologies) and growth factors comprising L-glutamine,hydrocortisone hemisuccinate, lineolic acid, licithin, human serumalbumin, basic fibroblasts growth factor, epidermal growth factor,transforming growth factor, insulin and/or vitamin C. The seeded surfaceis then placed in an incubator and grown at 37° C. in the presence ofhumidified air comprising 5% CO₂. As to remove any residue DMSO and/orother solvents that may be present in the cryogenic solution, the growthmedium may be changed every 24 to 48 hours following initiation of themonoculture. After which time, the growth medium may be changed every 48to 72 hours.

Other means of obtaining the initial fibroblasts may also be employed.For instance, fibroblasts may be isolated from neonatal foreskinretrieved from circumcision using the techniques detailed in co-pendingU.S. application Ser. No. 14/597,796.

When the fibroblast monolayer reaches approximately 80 to 90%confluence, the surface is transferred to a hood and the growth mediumremoved. The fibroblasts monolayer is then washed with a buffersolution, such as phosphate buffer solution without calcium ormagnesium. A sufficient volume of a cell detachment solution to promotedetachment of the fibroblasts from the second surface is then added. Forinstance, detachment of the fibroblasts may be promoted by adding 1 mlof Accutase Cell Detachment Solution, manufactured by Innovative CellTechnologies, Inc, per 25 cm² of growth area. The fibroblast cells arethen incubated in the cell detachment solution at 37° C. until all thecells have detached. A homogenous suspension of cells is then obtainedby mixing and the fibroblasts suspension is seeded onto cell free areaswithin the keratinocyte monolayer. The seeded culture is then returnedto the incubator. The co-culture is then grown in the keratinocytegrowth medium until 80 to 95% confluence is achieved.

A monolayer of co-cultured cells may also be achieved by culturingkeratinocytes in the keratinocyte growth medium until approximately 50%confluence is achieved. Cell free areas on the surface may then beseeded with the cultured fibroblasts suspension. For example, aco-culture in a T175 cm² flask would be overlayed with 1.5 ml offibroblast suspension generated from a confluent T75 cm² flask offibroblasts dissociated using 3 ml of Accutase. The co-culture may thenbe grown in the incubator until approximately 80 to 95% confluence isachieved.

The co-culture of cells may be stressed to provide a therapeutic serumsuitable for use in a cosmetic preparation. Stressing the co-culture maybe achieved by selectively removing nutrients, growth factors and/orother favorable conditions. The stress need not be severe. Accordingly,sufficient stress may be induced by removing all or a portion of thegrowth factors while maintaining nutrient levels. Growth factors may beremoved by extracting the keratinocyte growth medium from the surfaceand rinsing the co-culture monolayer twice with a sufficient volume of abuffer solution, such as phosphate buffer solution lacking calcium andmagnesium. As to ensure all growth factors are removed, the co-culturemay be incubated for a period of time in a collection medium that isadded to the surface and then discarded prior to serum collection. Forinstance, growth factors may be removed prior to serum collection byadding approximately 5.0 ml of a collection medium per 55 cm² of growtharea and incubating for approximately six hours.

The collection medium may comprise a minimum essential medium withEarl's salt and have the nutrients of the keratinocyte growth medium.

After removal of the growth factors, a sufficient volume of freshcollection medium is added, and the surface returned to the incubatorfor a sufficient period of time to produce a conditioned medium form thecollection medium. For example, incubating the co-culture inapproximately 10.0 ml of fresh collection medium per 55 cm² of growtharea for approximately 48 hours may be sufficient to produce aconditioned medium from the collection medium. After incubating for asufficient period of time, approximately 50% of the collection medium isremoved and replaced with an approximately equal amount of freshcollection medium. The co-culture is then incubated for approximately 48hours to produce more conditioned medium. After which time, all of theconditioned medium is removed.

The co-culture is then allowed to recover by removing the stress andincubating for a period of time. For instance, incubating in thepresence of approximately 10.0 ml per 55 cm² of growth area of thekeratinocyte growth medium for approximately 24 to 72 hours may providesufficient recovery. During recovery, the co-culture may be refreshed byseeding fresh cells of one or more of the cultures onto the monolayer.

After recovering, serum collection is repeated.

The process of the serum collection and recovery may be repeated untilthe co-cultures no longer produce serum of the desired quality. Forinstance, three passes may be utilized. The quality of serum may beginto degrade when one or more of the cultures used to initially establishthe co-culture reach 80% of their life expectancy as defined by themaximum number of population doublings.

The conditioned medium collected may be filtered using a suitablefilter, such as a 0.45 μm Millipore filter. The serum collected fromfiltering the conditioned medium may be tested for sterility, virologyand/or stability factors. Depending on the intended use of the serum,such testing may not be necessary.

The therapeutic serum collected may be incorporated into a suitabledelivery medium. For instance, a cosmetic base incorporating thetherapeutic serum may provide therapeutic cosmetic preparation useful asa recovery cream, moisturizer, neck cream, eye cream and/or facialcream. After combining the therapeutic serum to the base, antioxidants,and/or fragrances may be added to the cosmetic preparation. The cosmeticpreparation may comprise approximately 58 to 77 percent by mass of base,approximately 2 to 5 percent by mass of a moisturizer, approximately 9to 33 percent by mass of the serum, 3 to 14 percent by mass ofantioxidants, and approximately 0 to 0.01 percent by mass of fragrance.Other amounts and/or ingredients collectively providing a cosmeticallysuitable preparation may be utilized in combination with the serum.

In an exemplary embodiment, a facial cream cosmetic preparation may beprepared by adding 2.35 mass percent super low molecular weighthyaluronic acid added to 58.92 mass percent base. The base andhyaluronic acid combination may then be slowly mixed until homogenous.After which, 29.45 mass percent serum may be added to the base andhyaluronic acid mixture and slowly mixed until a homogenous mixture isobtained. Then 5.89 mass percent vitamin C may be slowly mixed in toprovide a homogenous mixture. After providing a homogenous mixtureincluding vitamin C, 2.94 mass percent vitamin E may be slowly mixed into provide a homogenous mixture. Then 0.44 mass percent vitamin A may bemixed in to provide a final homogenous vitamin-base-moisturizer-serumcomposition. To this composition, 0.01 mass percent fragrance may bemixed in to provide a final homogenous cosmetic preparation.

In an exemplary embodiment, an eye cream cosmetic preparation may beprepared by combining 61.83 mass percent base with 30.90 mass percentserum and slowly mixing until a homogenous mixture of base and serum isobtained. To this mixture, 3.707 mass percent super low molecular weighthyaluronic acid may be slowly mixed in to provide a homogenous mixture.Then, 3.09 mass percent vitamin E may be slowly mixed in to provideanother homogenous mixture. To this mixture, 0.463 mass percent vitaminA may be slowly mixed in to provide a homogenousvitamin-base-moisturizer-serum composition. To this composition, 0.01mass percent fragrance may be slowly mixed in to provide a finalhomogenous cosmetic preparation.

In an exemplary embodiment, a neck cream cosmetic preparation may beprepared by combining 67.243 mass percent base with 16.810 mass percentserum, and mixing slowly to provide a homogenous mixture of base andserum. To this mixture 4.203 mass percent super low molecular weighthyaluronic acid may be slowly mixed in to provide a homogenous mixture.Then, 7.985 mass percent vitamin C may be slowly mixed in to provide ahomogenous mixture. To this homogenous mixture, 3.329 mass percentvitamin E may be slowly mixed in to provide another homogenous mixture.After which, 0.42 mass percent vitamin A may be slowly mixed in toprovide a homogenous vitamin-base-moisturizer-serum composition. To thiscomposition, 0.01 mass percent fragrance may be mixed in to provide afinal homogenous cosmetic preparation.

In an exemplary embodiment, a moisturizer cream cosmetic preparation maybe prepared by combining 76.100 mass percent base with 9.605 masspercent serum and mixing slowly until a homogenous base-serum mixture isobtained. To this mixture, 3.42 mass percent super low molecular weighthyaluronic acid may be slowly mixed to provide another homogenousmixture. To this mixture, 6.60 mass percent vitamin C may be slowlymixed in to provide a homogenous mixture. After which, 3.805 masspercent vitamin E may be slowly mixed in to provide a homogenousmixture. Then, 0.46 mass percent vitamin A may be slowly mixed in toprovide a homogenous vitamin-base-serum-moisturizer composition. To thiscomposition, 0.01 mass percent fragrance may be mixed in to provide acosmetic preparation.

In an exemplary embodiment, a recovery cream cosmetic preparation may beprepared by combining 66.67 mass percent base with 33.33 mass percentserum and mixing slowly until homogenous.

While the present invention has been described herein with respect tothe exemplary embodiments, it will become apparent to one of ordinaryskill in the art that many modifications, improvements andsub-combinations of the various embodiments, adaptations and variationscan be made to the invention without departing from the spirit and scopethereof. Accordingly, the presented embodiment should not be construedas limiting the scope of this disclosure or the accompanying claims.

Furthermore, it should be appreciated that “first” and “second” as usedin claims is merely to reference that one (first) precedes another(second) and/or to distinguish similar components from one another. Itshould also be appreciated that though examples presented above may haveincluded only two cultures, this was solely for purposes of illustrationand in no way intended to limit the scope of this disclosure or theclaims. As such, a “first culture” may be a first, second, third, etc.culture. Likewise, the “second culture” may be any culture addedsubsequent to the “first culture”. Accordingly, if the “first culture”is the third culture added, then the “second culture” may be a fourth,fifth, sixth, etc. culture.

What is claimed:
 1. A method of producing a serum, comprising the stepsof: (a) seeding a first surface with a first culture of cells; (b)growing the first culture to a monolayer of less than 100% confluence inthe presence of a first growth medium, to provide at least one cell freearea on the first surface, the first growth medium comprising nutrientsand at least one growth factor, wherein the first growth medium promotesproliferation of the first culture; (c) seeding a second culture ontothe at least one cell free area of the first surface, the second culturecomprising cells different from those of the first culture; (d) growingthe first and second culture to less than 100% confluence in thepresence the second growth medium, the second growth medium comprisingof nutrients and at least one growth factor, wherein the second growthmedium promotes proliferation of the second culture; (e) after growingthe first and second cultures to less than 100% confluence, replacingthe second growth medium with a collection medium, the collection mediumlacking at least one of the growth factors of the second growth medium;(f) maintaining the first and second cultures for a period of time inthe presence of the collection medium to produce a conditioned medium;and (g) collecting at least a portion of the conditioned medium afterthe period of time, said conditioned medium comprising a therapeuticserum.
 2. The method of claim 1, wherein the first culture is grown to amonolayer of approximately 80 to 90% confluence, before seeding thesecond culture onto the at least one cell free area of the firstsurface.
 3. The method of claim 1, further comprising: before seedingthe second culture onto the first surface, increasing the cell freesurface area of on the first surface by creating voids in the monolayerof the first culture.
 4. The method of claim 3, wherein creating voidsin the monolayer of the first culture comprises mechanically detaching aportion of the monolayer of the first culture.
 5. The method of claim 3,wherein creating voids in the monolayer of the first culture comprises:treating the monolayer of the first culture with a detachment solution;after cells within the monolayer of the first culture begin to ball,removing the detachment solution; and squirting the monolayer of thefirst culture with the detachment solution.
 6. The method of claim 5,wherein the detachment solution comprises a proteolytic enzyme and acollagenolytic enzyme.
 7. The method of claim 1, wherein the nutrientsof the first growth medium are provided by a basal medium.
 8. The methodof claim 1, wherein the growth factors of the first growth mediumcomprise at least one of bovine pituitary extract, L-glutamine,hydrocortisone hemisuccinate, transforming growth factor, insulin,epinephrine, and ApoTransferrin.
 9. The method of claim 1, wherein thesecond growth medium comprises the nutrients and at least one growthfactor of the first medium.
 10. The method of claim 1, wherein thenutrients of the second growth medium are provided by a basal medium.11. The method of claim 1, wherein the growth factors of the secondgrowth medium comprise at least one of L-glutamine, hydrocortisonehemisuccinate, lineolic acid, lecithin, human serum albumin, basicfibroblasts growth factor, epidermal growth factor, transforming growthfactor, insulin, and vitamin C.
 12. The method of claim 1, furthercomprising growing the second culture on a second surface to less than100% confluence before seeding onto the first surface.
 13. The method ofclaim 1, wherein the collection medium comprises the nutrients of atleast one of the first growth medium and the second growth medium. 14.The method of claim 1, further comprising: after collecting at least aportion of the conditioned medium, replacing the conditioned medium witha recovery medium; and culturing the first culture and second culture inthe recovery medium for a recovery period of time.
 15. The method ofclaim 14, wherein the recovery medium comprises: the growth factors ofat least one of the first growth medium and the second growth medium;and the nutrients of at least one of the first growth medium and thesecond growth medium.
 16. The method of claim 1, wherein the secondculture comprises cells having a faster growth rate than those of thefirst culture.